During early embryogenesis, the embryo is divided into two major lineages: the pluripotent inner cell mass and the trophoblast. The pluripotent inner cell mass subsequently generates all three germ layers, which are capable of further differentiation into terminally differentiated tissue specific cells.
Embryonic stem cells are self-renewing cells derived from the inner cell mass of the embryo at the blastocyst stage and the embryonic stem cells possess the potential to differentiate into any one of the three germ lineages (i.e., are pluripotent). The pluripotent stem cells have practical applications in clinical settings such as in the areas of regenerative medicine and chemotherapy.
There is a need in the art for methods and compositions that are capable of modulating the development of pluripotent stem cells, specifically the differentiation of pluripotent stem cells along a specific preferred lineage.
The evolutionary conserved Wnt signalling pathway controls many events in embryogenesis and also has a central role in tumorigenesis. The Wnt signalling pathway is a signalling cascade of a collection of proteins which regulates the phosphorylation and degradation of β-catenin, thereby regulating expression of β-catenin-dependent genes.
The Wnt genes belong to a family of proto-oncogenes expressed in several species ranging from invertebrates to vertebrates. These genes encode over twenty cysteine-rich, secreted glycoproteins that activate the Wnt signalling pathway by binding to Frizzled (Fz) receptors found on target cells.
Binding of Wnt ligands to Frizzled receptors activates the Dishevelled (Dsh/Dvl1) protein, allowing it to inhibit the activity of a multiprotein complex comprising β-catenin, Axin-adenomatous polyposis coli (APC) and glycogen synthase kinase (GSK)-3β. Inhibition of the β-catenin/APC/GSK-3β complex prevents phosphorylation of β-catenin by GSK-3β. Phosphorylated β-catenin is targeted for ubiquitin mediated degradation by the proteosome, and therefore Wnt binding to the Frizzled receptor results in β-catenin accumulation in the cytoplasm.
Stabilized β-catenin translocates into the nucleus and binds to members of T-cell factor (Tcf)/Lymphoid enhancing factor (Lef) family of proteins, resulting in the transcription of Wnt target genes.
Reya et al (2003) Nature 423(6938):409-14 discloses a role for Wnt signalling in self-renewal of haematopoietic stem cells (non-pluripotent stem cells). Thus, activation of the Wnt signalling pathway in haematopoietic stem cells maintains pluripotency in these cells.
In contrast, a number of documents in the prior art suggest that activation of Wnt signalling in non-pluripotent stem cells leads to differentiation.
Thus, Lako et al (2001, Mechanisms of Development 103, 49-59) describes a role for Wnt signalling in enhancing differentiation of embryoid bodies. Cells in embryoid bodies are multipotent, but are not pluripotent, i.e., are not capable of giving rise to all three germ layers. Specifically, Lako discloses that activation of Wnt signalling by over-expression of Wnt3 results in haematopoietic differentiation of embryoid bodies.
International Patent Publication WO 2004/113513 describes the use of Wnt polypeptides in the modulation of proliferation or differentiation of a population of adult stem cells, specifically haematopoietic CD45+Sca1+ stem cells.
WO 2005/052141 provides a number of methods for inducing or inhibiting differentiation of foetal lung stem cells. One particular method disclosed in WO2005/052141 involves in vitro up-regulation of the Wnt pathway in foetal lung stem cells, which results in inhibition of differentiation.
Despite these teachings of the role of Wnt signalling in the induction of differentiation in multipotent stem cells, the Wnt signalling appears to have an opposite role in the regulation of the choice between pluripotency/differentiation in pluripotent embryonic stem cells. Thus, activation of Wnt signalling in embryonic stem cells appears to result in maintenance of the pluripotent state and inhibition of differentiation.
Thus, Sato et al (2004 Nature Medicine 10: 55-63) discloses that activation of Wnt signalling in human and mouse embryonic stem cells leads to maintenance of pluripotency of such stem cells and inhibition of differentiation. In support of this, US 2004/0014209 A1 discloses that inhibition of Wnt signalling pathway plays a role in the stimulation of differentiation of stem cells, including embryonic stem cells, into cardiac cells.